MNTF peptides and compositions and methods of use

ABSTRACT

The present invention is directed to novel peptides and compositions capable of modulating viability and growth in neuronal cells, and to methods of modulating neuronal cell viability and growth employing the novel peptides and compositions of the invention. In one aspect, the invention is directed to novel peptide analogues of motoneuronotrophic factor 1 containing either a “WMLSAFS” or “FSRYAR domain,” which is sufficient for neurotrophic or neurotropic function.

RELATED APPLICATIONS

The present application claims the benefit of U.S. provisionalapplication No. 60/441,772 filed Jan. 21, 2003, which is incorporatedherein by reference in its entirety.

BACKGROUND

Neuronotrophic factors (NTFs) are a specialized group of proteins whichfunction to promote the survival, growth, maintenance, and functionalcapabilities of selected populations of neurons. Recent studies havedemonstrated that neuronal death occurs in the nervous systems ofvertebrates during certain periods of growth and development. However,the addition of soluble neuronal trophic factors from associated targettissues serves to mitigate this phenomenon of neuronal death. Thefollowing citations discuss neuronal trophic factors and theirdisclosures are hereby incorporated by reference: Chau, R. M. W., etal., Neuronotrophic Factor, 6 Chin. J Neuroanatomy 129 (1990); Kuno, M.,Target Dependence of Motoneuronal Survival: The Current Status, 9Neurosci. Res. 155 (1990); Bard, Y. A., Trophic Factors and NeuronalSurvival, 2 Neuron 1525 (1989); Oppenheim, R. W., The NeurotrophicTheory and Naturally Occurring Motoneuron Death, 12 TINS 252 (1989);Bard, Y. A., What, If Anything, is a Neurotrophic Factor?, 11 TINS 343(1988); and Thoenen, H., and Edgar, D., Neurotrophic Factors, 229Science 238 (1985).

In the vertebrate neuromuscular system, the survival of embryonicmotoneurons have been found to be dependent upon specific trophicsubstances derived from the associated developing skeletal muscles.Skeletal muscles have been shown, by both in vivo and in vitro studies,to produce substances which are capable of enhancing the survival anddevelopment of motoneurons by preventing the embryonic motoneurons fromdegeneration and subsequent, natural cellular death. See O'Brian, R. J.and Fischbach, G. D., Isolation of Embryonic Chick Motoneurons and TheirSurvival In Vitro, 6 J. Neurosci. 3265 (1986); Hollyday, M. andHamburger, V., Reduction of the Naturally Occurring Motor Neuron Loss byEnlargement of the Periphery, 170 J. Comp. Neurol. 311 (1976), whosedisclosures are incorporated herein by reference. Similarly, severalinvestigators have reported that chick and rat skeletal muscles possesscertain trophic factors which can prevent the natural cellular death ofembryonic motoneurons both in vivo and in vitro. See McManaman, J. L.,et al., Purification of a Skeletal Muscle Polypeptide Which StimulatesCholine Acetyltransferase Activity in Cultured Spinal Cord Neurons, 263J. Biol. Chem. 5890 (1988); Oppenheim, R. W., et al., Reduction ofNaturally Occurring Motoneuron Death In Vitro by a Target DerivedNeurotrophic Factor, 240 Science 919 (1988); and Smith, R. G., et al.,Selective Effects of Skeletal Muscle Extract Fractions on MotoneuronsDevelopment In Vivo, 6 J. Neurosci. 439 (1986), whose disclosures areincorporated herein by reference.

In addition, a polypeptide has been isolated from rat skeletal musclewhich has been found to selectively enhance the survival of embryonicchick motoneurons in vivo, as well the activity of cholineacetyltransferase in these motoneurons. This polypeptide has been namedCholine Acetyltransferase Development Factor (CDF) and its biologicalfunction has been demonstrated to be different from other trophicfactors such as Nerve Growth Factor (NGF), Ciliary Ganglion NeurotrophicFactor (CNTF), Brain-Derived Neurotrophic Factor (BDNF), and RetinalGanglion Neurotrophic Factor (RGNTF). See Levi-Montalcini, R.,“Developmental Neurobiology and the Natural History of Nerve GrowthFactor,” 5 Ann. Rev. Neurosci. 341 (1982); Varon, S., et al., GrowthFactors. In: Advances in Neurology, Vol. 47: Functional Recovery inNeurological Disease, Waxman, S. G. (ed.), Raven Press, New York, pp.493–521 (1988); Barde, Y. A., Trophic Factors and Neuronal Survival, 2Neuron 1525 (1989); Chau, R. M. W., et al., The Effect of a 30 kDProtein from Tectal Extract of Rat on Cultured Retinal Neurons, 34Science in China, Series B, 908 (1991), whose disclosures areincorporated herein by reference.

The isolation and characterization of two motoneuronotrophic factorsfrom rat muscle tissue having apparent molecular weights of 35 kD and 22kD were reported by Chau et al. See Chau, R. M. W., et al., MuscleNeuronotrophic Factors Specific for Anterior Horn Motoneurons of RatSpinal Cord. In: Recent Advances in Cellular and Molecular Biology, Vol.5, Peeters Press, Leuven, Belgium, pp. 89–94 (1992), the disclosure ofwhich is hereby incorporated by reference. The 35 kD protein has beendefined by Dr. Chau as motoneuronotrophic factor 1 (MNTF1) and theapparent 22 kD protein as motoneuronotrophic factor 2 (MNTF2). These twotrophic factors have been demonstrated in vitro to support the growthand/or regeneration of both isolated anterior horn motoneurons andspinal explants of rat lumber spinal cord.

Subsequently, in 1993, Chau et al reported immunological screening oflambda gt11 clones from a human retinoblastoma cDNA library using amonoclonal antibody to MNTF1 as an immunoprobe. Immunoblots of extractsfrom an immunopositive clone stained an MNTF1 protein having an apparentmolecular weight of 55 kD See Chau, R. M. W., et al., Cloning of Genesfor Muscle-Derived Motoneuronotrophic Factor 1 (MNTF1) and Its Receptorby Monoclonal Antibody Probes, (abstract) 19 Soc. for Neurosci. part 1,252 (1993), the disclosure of which is hereby incorporated by reference.An extract containing the cloned human MNTF1 was shown to havebiological activity similar to that of the “native” MNTF1 protein inthat it supported the in vitro growth of rat anterior horn motoneurons.

More recently, U.S. Pat. No. 6,309,877 disclosed a family ofneuronotrophic factors which possess the ability to exert a trophiceffect on motoneurons. The motoneurotrophic factors were isolated,nucleic acid sequences encoding these factors were cloned and expressed,and both the nucleic acid and polypeptide sequences were provided. Inparticular, recombinant proteins MNTF1–F3 and MNTF1–F6 encoded by 1443and 972 base pair inserts, respectively, were expressed as either fusionproteins or purified fragments. The isolated factors and the expressed,recombinant factors, were capable of inducing the continued viabilityand neurite outgrowth of motoneurons. Therefore, these factors have beenclassified as “motoneuronotrophic factors” or “MNTFs.”

The MNTF1–F6 clones reported in U.S. Pat. No. 6,309,877 encode a 33amino acid fragment of MNTF1. Recombinant protein containing thissequence reacted with monoclonal antibody to MNTF1, maintainedmotoneuron viability, increased neurite outgrowth, reduced celldeath/apoptosis and supported the growth and “spreading” of motoneuronsinto giant, active neurons with extended growth cone-containing axons.Consequently, the following studies were conducted to determine if apeptide comprising a “minimal” active site can be synthesized, whichstill retains the biological activity of this MNTF1 fragment.

SUMMARY OF THE INVENTION

The present invention is directed to novel peptides and compositionscontaining portions of the MNTF-molecule that are useful for modulatingthe viability and proliferation of neuronal cells, thereby providing forneurotrophic peptides that can be readily synthesized.

In particular, the present invention is directed to a novel proteindomain of general significance to the actions of motoneuronotrophicfactors, which has been identified and mapped to two short overlappingsubsequences in the MNTF1 molecule. These heretofore unrecognizedprotein domains, which is designated herein the “WMLSAFS” and “FSRYAR”domains, are sufficient to modulate the viability and proliferation ofneuronal cells. Moreover, truncated MNTF1 species encompassing thesedomain are themselves sufficient to stimulate the growth ofmotoneuron/neuroblastoma cell hybrids in cell proliferation assays.

In one aspect, then, the invention is directed to purified and isolatedMNTF peptide analogues comprising the WMLSAFS (SEQ ID NO: 3) or FSRYAR(SEQ ID NO: 2) domains and to molecules that mimic its structure and/orfunction, useful for inducing or modulating the viability and growth ofa neuronal cell. 1. Particular embodiments of such MNTF peptideanalogues are disclosed herein as SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

The present invention also relates to compositions and methods formodulating the viability and/or growth of a neuronal cells byadministering the MNTF peptide analogues in vitro to cell cultures or invivo to an individual suffering from a nerve injury or neurodegenerativedisorder, in order to promote cell proliferation or stabilizeinappropriate cell death, and/or in either case to restore normal cellbehavior. The present invention is also directed to the use of MNTFpeptide analogues for its antiproliferative effects on non-neuronalcells, particularly its use as an antifibrotic or anti-inflammatoryagent in wound healing

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription and accompanying drawings, where:

FIG. 1 shows amino acid sequences of various MNTF1 peptides;

FIG. 2 shows percent increases in proliferation of VSC4.1 cells byMNTF1–F6 33mer and its peptide derivatives at various doses;

FIG. 3 also shows percent increases in proliferation of VSC4.1 cells byhighly purified (GLPg) MNTF1–F6 33mer and its peptide derivatives atvarious doses;

FIG. 4 shows percent increases in proliferation of VSC4.1 cells byMNTF1–F6 33mer and additional peptide derivatives at various doses; and

FIG. 5 shows selective reinnervation of target muscle cells by motorneurons treated with MNTF 6mer at various doses.

DETAILED DESCRIPTION

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of skill in the art.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. Standard reference works settingforth the general principles of recombinant DNA technology includeSambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Planview, N.Y. (1989); McPherson,M. J., Ed., Directed Mutagenesis: A Practical Approach, IRL Press,Oxford (1991); Jones, J., Amino Acid and Peptide Synthesis, OxfordScience Publications, Oxford (1992); Austen, B. M. and Westwood, O. M.R., Protein Targeting and Secretion, IRL Press, Oxford (1991). Anysuitable materials and/or methods known to those of skill can beutilized in carrying out the present invention; however, preferredmaterials and/or methods are described herein.

Overview

The isolation and characterization of two mononeuronotrophic factors(MNTF1 and MNTF2) from rat muscle tissues as well as the subsequentcloning of a recombinant MNTF1–F6 gene derived from a humanretinoblastoma cDNA library, is described in U.S. Pat. No. 6,309,877 (aswell as co-pending U.S. patent application Ser. Nos. 09/989,481,08/928,862, filed Sep. 12, 1997, Ser. No. 08/751,225, filed Nov. 15,1996, and U.S. provisional patent application 60/026,792, filed on Sep.27, 1996; all of which are hereby incorporated by reference in theirentirety). The MNTF1–F6 gene sequence encodes a 33 amino acid sequencereferred to herein as SEQ ID NO:1.

The naturally occurring and recombinant MNTF1 polypeptides were shown toselectively enhance the survival in vitro of anterior horn motor neuronsisolated from rat lumbar spinal cord explants. Photomicrographs oftreated cultures exhibited neurite outgrowth of myelinated nerve fibersand a marked reduction in the growth of non-neuronal cells, e.g. glialcells and fibroblasts. Similarly, in vivo administration of MNTF1 tosurgically axotomized rat peripheral nerves resulted in a markedlyhigher percentage of surviving motor neurons than untreated controls,which could be blocked by co-administration of anti-MNTF1 monoclonalantibody.

Further beneficial effects of MNTF1 were demonstrated in rats subjectedto spinal cord hemi-section, repaired by a peripheral nerve autograftand implanted with MNTF1-containing gel sections in close proximity tothe nerve graft junctions with spinal cord. MNTF1 treated animalsexhibited greater numbers of surviving motor neurons, improved recoveryof motor and sensory function, reduced inflammatory response (fewerinfiltrating macrophages and lymphocytes), reduced collagen-containingscar tissue formation at the site of the graft, normal Schwann cellmorphology and normal myelinated and non-myelinated nerve fiberformation.

The efficacy of MNTF in the treatment of neurogenerative disease wasalso demonstrated in the wobbler mouse animal model. Wobbler mice carryan autosomal double-recessive gene mutation that leads to theprogressive degeneration of spinal and brain stem motor neurons.Approximately three weeks post partum, wobbler mice begin to develop the“wobbling” symptomology (Stage 1) with concomitant degeneration ofcervical motor neurons leading to both the wasting of the muscle of theforelimbs and an inability to extend the digits and claws. By threemonths of age the pathologic symptomology progresses to stage 4, with a“clumping together” of all associated joints in the forelimbs, e.g., thewrist, elbow and shoulder joints, as well as an extensive loss of bodyweight and chronic fatigue. However, most wobbler mice die prior toreaching three months of age. Implantation of MNTF1-containing gelsections between the trapezius and rhomboid muscles and the C7-T3 regionof the spinal cord delayed the progression of symptoms in wobbler mice,resulting in a general improvement in life span, health, respiration,body weight, strength of forelimbs as well as reduced vacuolation andchromatolysis of their cervical motor neurons compared to the controlgroup.

Two previously unrecognized overlapping domains within the MNTF1–F6molecule that appear to be sufficient for the known biologicalactivities of MNTF1 have now been identified. Each of these domains,designated herein as the “WMLSAFS” and “FSRYAR” domains, are sufficientto stimulate the proliferation of motor neuron derived cell lines in amanner similar to the MNTF1–F6 33-mer. Similarly, the “FSRYAR” domain issufficient to direct selective reinnervation of muscle targets by motorneurons in vivo in a manner similar to the MNTF1–F6 33-mer. In addition,the “FSRYAR” domain provides an antigenic epitope sufficient to raiseantibody that recognizes any MNTF peptide containing the “FSRYAR”sequence, including the MNTF1–F6 33-mer.

As those of skill familiar with the art and the present invention willappreciate, sequences comprising the WMLSAFS and/or FSRYAR domain(s)provide MNTF peptide analogues for use in selectively modulating theviability and morphology of neuronal cells versus non-neuronal cells invitro and in vivo. Moreover, compounds and compositions, which arecapable of binding to the WMLSAFS and/or FSRYAR domain(s), provideagents for use in the detection and/or modulation of MNTF1 activity intarget cells and tissues.

Peptides

As used herein, the term “WMLSAFS domain” or “FSRYAR domain” refers to apolypeptide domain demonstrated herein to be sufficient for theselective maintenance and axonal regeneration of neuronal cells, and topeptides and/or molecules capable of mimicking their structure and/orfunction. Preferred embodiments of the present invention comprise apeptide having the amino acid sequence: FSRYAR [SEQ ID NO:2] or WMLSAFS[SEQ ID NO:3], as well as functional equivalents thereof.

By “functional equivalent” is meant a peptide possessing a biologicalactivity substantially similar to that of the WMLSAFS and/or FSRYARdomain(s), and is intended to include “fragments”, “variants”,“analogs”, “homologs”, or “chemical derivatives” possessing suchactivity or characteristic. Functional equivalents of the WMLSAFS and/orFSYAR domain(s), then, may not share an identical amino acid sequences,and conservative or non-conservative amino acid substitutions ofconventional or unconventional amino acids are possible.

As used herein, the terms “biologically active peptide” and“biologically active fragment” refer to a peptide or polypeptide inaccordance with the above description of motoneuronotrophic factors(MNTF) wherein the MNTF exhibits a protective effect on motor neuronsand/or a proliferative effect on motor neuron derived cell lines.

The sequence of amino acid residues in a protein or peptide comprisingthe MNTF peptide analogues of the present invention are designatedherein either through the use of their commonly employed three-letterdesignations or by their single-letter designations. A listing of thesethree-letter and one-letter designations may be found in textbooks suchas Biochemistry, Second Edition, Lehninger, A., Worth Publishers, NewYork, N.Y. (1975). When the amino acid sequence is listed horizontally,the amino terminus is intended to be on the left end whereas the carboxyterminus is intended to be at the right end.

Reference herein to “conservative” amino acid substitution is intendedto mean the interchangeability of amino acid residues having similarside chains. For example, glycine, alanine, valine, leucine andisoleucine make up a group of amino acids having aliphatic side chains;serine and threonine are amino acids having aliphatic-hydroxyl sidechains; asparagine and glutamine are amino acids having amide-containingside chains; phenylalanine, tyrosine and tryptophan are amino acidshaving aromatic side chains; lysine, arginine and histidine are aminoacids having basic side chains; and cysteine and methionine are aminoacids having sulfur-containing side chains. Interchanging one amino acidfrom a given group with another amino acid from that same group would beconsidered a conservative substitution. Preferred conservativesubstitution groups include asparagine-glutamine, alanine-valine,lysine-arginine, phenylalanine-tyrosine and valine-leucine-isoleucine.

It will be appreciated by those of skill that the precise chemicalstructure of peptides comprising the various MNTF peptide analogues willvary depending upon a number of factors. For example, a givenpolypeptide may be obtained as an acidic or basic salt, or in neutralform, since ionizable carboxyl and amino groups are found in themolecule. For the purposes of the invention, then, any form of thepeptides comprising the WMLSAFS and/or FSRYAR domain(s), which retainthe biological activity of the MNTF1 33mer peptide is intended to bewithin the scope of the present invention.

MNTF1–F6 33-mer

In U.S. Pat. No. 6,309,877, there is provided a polypeptide (referred totherein as SEQ ID NO: 4) having the following amino acid sequence:

-   -   LGTFWGDTLNCWMLSAFSRYARCLAEGHDGPTQ [SEQ ID NO:1]

Recombinant protein containing this sequence reacted with monoclonalantibody to MNTF-1, maintained motoneuron viability, increased neuriteoutgrowth, reduced motoneuron cell death/apoptosis and supported thegrowth and “spreading” of motoneurons into giant, active neurons withextended growth cone-containing axons. The MNTF1 33-mer, referred toherein as SEQ ID NO: 1, was synthesized by solid phase synthesis andserved as positive control in cell proliferation assays, as described inthe examples below. The linear 33-mer was found to be effective atincreasing the proliferation of motoneuron/neuroblastoma cells, whereasa cyclized version of the peptide was less effective.

The present invention includes peptide analogues of MNTF1 that retainthe ability of MNTF1 to promote the survival and maintenance of motorneurons. An MNTF peptide analogue in accordance with the presentinvention is typically 6 to 32 amino acids in length and contains atleast one of two amino acid sequences, namely the WMLSAFS domain (SEQ IDNO:3) corresponding to amino acid residues 12 to 18 of SEQ ID NO:1, orthe FSRYAR domain (SEQ ID NO:2) corresponding to amino acid residues 17to 22 of SEQ ID NO:1. Preferred embodiments of the MNTF peptide analogueinclude a fragment of six to 32 consecutive amino acid residues of SEQID NO:1.

In alternative embodiments the amino sequence of the motoneuronotrophicfactor peptide analogue is at least 70% identical to nine to 32consecutive amino acid residues of SEQ ID NO: 1, at least 80% identicalto eight to 32 consecutive amino acid residues of SEQ ID NO: 1 and mostpreferably, a least 90% identical to seven to 32 consecutive amino acidresidues of SEQ ID NO: 1 as determined by BLAST analysis.

To compare a polypeptide sequence with the corresponding SEQ ID NO:1fragment, a global alignment of the sequences can be performed using theBLAST programs publicly available through the National Center forBiotechnology Information (on the World Wide Web at ncbi.nlm.nih.gov).Prior to performing a global alignment, SEQ ID NO:1 can be submitted toGenBank. Default parameters provided by the National Center forBiotechnology Information can be used for a global alignment.

6-mer

In a particularly preferred embodiment, there is provided a peptidehaving the following amino acid sequence:

-   -   FSRYAR    -   Phe-Ser-Arg-Tyr-Ala-Arg [SEQ ID NO:2]        corresponding to amino acid residues 17–22 of SEQ ID NO:1, which        was found to be sufficient to increase cell proliferation of        motor neuron/neuroblastoma cells. This portion of the MNTF-1        molecule will be referred to hereinafter as the “FSRYAR” domain.        7-mer

In another preferred embodiment, there is provided a peptide having thefollowing amino acid sequence:

-   -   WMLSAFS    -   Trp Met Leu Ser Ala Phe Ser [SEQ ID NO:3]        corresponding to amino acid residues 12–18 of SEQ ID NO:1. This        7 amino acid fragment of MNTF1 overlaps the FS residues of the        FSRYAR domain. The peptide was also found to be a potent        stimulator of motor neuron/neuroblastoma cells in vitro over a        broad range of dosage levels. This portion of the MNTF-1        molecule will be referred to hereinafter as the “WMLSAFS”        domain.        10-mer

In another preferred embodiment, there is provided a peptide having thefollowing amino acid sequence:

-   -   MLSAFSRYAR    -   Met Leu Ser Ala Phe Ser Arg Tyr Ala Arg [SEQ ID NO:4]        corresponding to amino acid residues 13–22 of SEQ ID NO:1. This        MNTF fragment includes most of the “WMLSAFS” domain as well as        the entire FSRYAR domain. The 10mer was at least as effective        the full-length MNTF 33mer at stimulating motor        neuron/neuroblastoma cells in vitro at concentrations as low as        0.01 μg/ml.        11-mer

In another preferred embodiment, there is provided a peptide having thefollowing amino acid sequence:

-   -   FSRYARCLAEG    -   Phe-Ser-Arg-Tyr-Ala-Arg-Cys-Leu-Ala-Glu-Gly [SEQ ID NO:5]        corresponding to amino acid residues 17–27 of SEQ ID NO:1. This        11-mer contains the FSRYAR domain and was also found to be        sufficient to increase cell proliferation of motor        neuron/neuroblastoma cells.        13-mer

In another preferred embodiment, there is provided a peptide having thefollowing amino acid sequence:

-   -   CWMLSAFSRYARC    -   Cys Trp Met Leu Ser Ala Phe Ser Arg Tyr Ala Arg Cys [SEQ ID        NO:6]        corresponding to amino acid residues 11 to 23 of SEQ ID NO:1.        This 13-mer contains both WMLSAFS and FSRYAR domains and was        also found to be sufficient to increase cell proliferation of        motor neuron/neuroblastoma cells. However, a cyclized version of        the 13-mer was not as effective at stimulating cell        proliferation in vitro.        21-mer

In another preferred embodiment, there is provided a peptide having thefollowing amino acid sequence:

-   -   MLSAFSRYARCLAEGHDGPTQ    -   Met Leu Ser Ala Phe Ser Arg Tyr Ala Arg Cys, Leu Ala Glu Gly His        Asp Gly Pro Thr Gln [SEQ ID NO:7]        corresponding to amino acid residues 13 to 33 of SEQ ID NO:1.        This 21-mer contains most of the “WMLSAFS” domain as well as the        entire FSRYAR domain and was also found to be sufficient to        increase cell proliferation of motor neuron/neuroblastoma cells.        MNTF Peptide Analogues

It is to be understood that within the scope of the present inventionare peptide analogues as described and identified herein in which one ormore amino acids are substituted with other amino acids. In a preferredalternative, the motoneuronotrophic factor peptide analogue contains oneor more conservative amino acid substitutions to a fragment of seven to32 consecutive amino acid residues of SEQ ID NO:1.

An MNTF peptide analogue within the scope of this invention can be analtered form of an MNTF1 peptide providing generally of course that theessential activity of the peptide remains substantially unchanged. Asused herein, the term “altered form” refers to a peptide that has beentreated to change its naturally occurring structure. An altered form canbe prepared, for example, by covalent modification of an MNTF1 peptidefragment, by crosslinking MNTF1 peptide fragment to an insoluble supportmatrix, or by crosslinking MNTF1 peptide fragment to a carrier protein.

An MNTF1 peptide analogue within the scope of this invention can be apeptide fragment that is antigenically related to an MNTF1 peptidefragment. Two peptides, which are antigenically related displayimmunological cross-reactivity. For example, antibodies to the firstpeptide also recognize the second peptide.

An MNTF1 peptide analogue within the scope of this invention can be afusion protein containing a MNTF1 peptide fragment attached to aheterologous protein. A heterologous protein has an amino acid sequencenot substantially similar to the MNTF1 peptide fragment. Theheterologous protein can be fused to the N-terminus or C-terminus of theMNTF1 peptide fragment. Fusion proteins can include, but are not limitedto, poly-His fusions, MYC-tagged fusions, Ig fusions and enzymaticfusion proteins, for example beta-galactosidase fusions. Such fusionproteins, particularly poly-His fusions, can facilitate the purificationof recombinant MNTF1 peptide fragments.

Peptidomimetics of WMLSAFS and/or FSRYAR domain peptide(s) are alsoprovided by the present invention, and can act as drugs for themodulation of neuronal cell viability and growth by, for example,blocking the function of proteins comprising the WMLSAFS and/or FSRYARdomain(s). Peptidomimetics are commonly understood in the pharmaceuticalindustry to include non-peptide drugs having properties analogous tothose of those of the mimicked peptide. The principles and practices ofpeptidomimetic design are known in the art and are described, forexample, in Fauchere J., Adv. Drug Res. 15: 29 (1986); and Evans et al.,J. Med. Chem. 30: 1229 (1987).

Peptidomimetics which bear structural similarity to therapeuticallyuseful peptides may be used to produce an equivalent therapeutic orprophylactic effect. Typically, such peptidomimetics have one or morepeptide linkages optionally replaced by a linkage, which may convertdesirable properties such as resistance to chemical breakdown in vivo.Such linkages may include —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—, —COCH₂—,—CH(OH)CH₂—, and —CH₂SO—. Peptidomimetics may exhibit enhancedpharmacological properties (biological half life, absorption rates,etc.), different specificity, increased stability, production economies,lessened antigenicity and the like which makes their use as therapeuticsparticularly desirable.

The rational design of WMLSAFS and/or FSRYAR domain mimetics or bindingmolecules, based on modeled (or experimentally determined) peptidestructure, may be carried out by those of skill, using known methods ofrational drug design. The goal of rational drug design is to producestructural analogs of biologically active polypeptides or targetcompounds. By creating such analogs, it is possible to fashion drugs,which are more active or stable than the natural molecules, which havedifferent susceptibility to alteration or which may affect the functionof various other molecules. In one approach, one would generate athree-dimensional structure for a target molecule, or a fragmentthereof. This could be accomplished by x-ray crystallography, computermodeling or by a combination of both approaches.

Methods of Making

It is understood that an MNTF peptide composition of the presentinvention may be made by a method that is well known in the art,including but not limited to chemical synthesis by solid phase synthesisand purification away from the other products of the chemical reactionsby HPLC, or production by the expression of a nucleic acid sequence(e.g., a DNA sequence) encoding a peptide or polypeptide comprising anMNTF peptide of the present invention in an in vitro translation systemor in a living cell. Preferably the MNTF peptide of the composition isisolated and extensively dialyzed to remove one or more undesired smallmolecular weight molecules and/or lyophilized for more ready formulationinto a desired vehicle. It is further understood that additional aminoacids, mutations, chemical modification and such like, if any, that aremade in a MNTF peptide component will preferably not substantiallyinterfere with receptor recognition of the MNTF docking sequence.

A peptide or polypeptide corresponding to one or more fragments of MNTF1of the present invention should generally be at least five or six aminoacid residues in length, and may contain up to about 7, about 8, about9, about 10, about 11, about 12, about 13, about 15, about 20 or about30 residues or so. A peptide sequence may be synthesized by methodsknown to those of ordinary skill in the art, such as, for example,peptide synthesis using automated peptide synthesis machines, such asthose available from Applied Biosystems (Foster City, Calif.). Theinvention further provides the synthesis and use of cyclic peptides suchas those derived from (SEQ ID NO:1) and (SEQ ID NO:6) as shown in Table1 below.

Covalent modifications can be introduced into a peptide by reactingtargeted amino acid residues with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues.Covalent modification of polypeptides using organic derivatizing agentsis well known to those of skill in the art. For example, cysteinylresidues can be reacted with α-haloacetates (and corresponding amines),such as chloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Histidyl residues can be derivatized byreaction with diethylpyrocarbonate at pH 5.5–7.0, or withpara-bromophenacyl bromide at pH 6 in 1 M sodium cacodylate. Lysinyl andamino terminal residues can be reacted with succinic or other carboxylicacid anhydrides. Arginyl residues can be modified by reaction with oneor several conventional reagents, among them phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Spectral labelscan be introduced into tyrosyl residues by reaction with aromaticdiazonium compounds or tetranitromethane; most commonly,N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosylspecies and 3-nitro derivatives, respectively. Carboxyl side groups(aspartyl or glutamyl) can be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions. Glutaminyl and asparaginyl residues canbe deamidated to the corresponding glutamyl and aspartyl residues. Othermodifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, 1983, Proteins: Structure and MoleculeProperties, W.H. Freeman & Co., San Francisco, pp. 79–86), acetylationof the N-terminal amine, and, in some instances, amidation of theC-terminal carboxyl groups.

The invention further provides the novel MNTF peptide analogues for usein assays and kits for assays, either in the free form or linked to acarrier molecule such as a protein or a solid particle, as well asmodified peptides linked to a label or tracer e.g. biotin or fluoresceinisothiocyanate.

Crosslinking of MNTF1 peptide fragment to a water-insoluble supportmatrix can be performed with bifunctional agents well known in the artincluding 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Bifunctional agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates can be employed for proteinimmobilization.

Crosslinking of an MNTF1 peptide fragment to a second protein, includinga second MNTF1 peptide fragment, can be performed using the bifunctionalreagents described herein. In another alternative, there is inserted aspacer, for example a dithiol group or a diamino group or multiples ofamino acid residues, e.g. glycine. The spacer may also be a homo- orhetero-bifunctional crosslinker, for example the heterobifunctionalcrosslinker N-(4-carboxy-cyclohexyl-methyl)-maleimide.

Antibodies to an MNTF1 peptide fragment can be prepared by methods thatare well known in the art (see, e.g., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988). A wide range of animal species canbe used for the production of antibodies. Typically the animal used forproduction of antibodies is a rabbit, a mouse, a rat, a hamster, aguinea pig and/or a goat. Antiserum can be used as is for variousapplications. Alternatively, the desired antibody fraction can bepurified by well-known methods such as affinity chromatography usinganother antibody, protein A and protein G chromatography, andchromatography using a peptide bound to a solid matrix.

Immunological cross-reactivity can be determined using standardimmunological assays well known in the art. For example enzyme linkedimmunosorbent assay (ELISA) can be performed by immobilizing an MNTF1peptide analogue onto the well surface of a microtiter plate, thencontacting the immobilized MNTF1 peptide analogue with antibodies to anMNTF1 peptide fragment. After washing to remove unbound andnon-specifically bound antibody, the bound antibody can be detected.Where the initial antibodies are linked to a detectable label, the boundantibody can be detected directly. Alternatively, the bound antibody canbe detected using a second antibody that has binding affinity for thefirst antibody, with the second antibody being linked to a detectablelabel.

Longer peptides or polypeptides, e.g a fusion protein, can be producedby standard recombinant DNA techniques. For example, a DNA fragmentencoding a MNTF1 peptide fragment can be cloned in a commerciallyavailable expression vector that already contains a heterologousprotein, with the result being MNTF1 peptide fragment fused in-frame tothe heterologous protein.

In certain embodiments, a nucleic acid encoding an MNTF peptide and/or acomponent described herein may be used, for example, to produce apeptide in vitro or in vivo for the various compositions and methods ofthe present invention. For example, in certain embodiments, a nucleicacid encoding an MNTF peptide is a component of, for example, a vectorin a recombinant cell. The nucleic acid may be expressed to produce apeptide or polypeptide comprising an MNTF peptide sequence. The peptideor polypeptide may be secreted from the cell, or as part of or withinthe cell.

Compositions

Pharmaceutical compositions in accordance with the present inventionpreferably comprise one or more of the MNTF1 peptide analogues of thepresent invention together with a pharmaceutically acceptable diluentand/or carrier. Suitable carriers/diluents are well known in the art andinclude saline or other sterile aqueous media, optionally includingadditional components such as buffer salts and preservatives, or sugars,starches, salts or mixtures thereof.

The pharmacological compositions of the present invention are preparedin conventional dosage unit forms by the incorporation of one or more ofthe MNTF peptide analogues with an inert, non-toxic pharmaceutical“carrier” moiety according to accepted methodologies, in a non-toxicconcentration sufficient to produce the desired physiological activityin a mammal and, in particular, a human subject. Preferably, thecomposition contains the active ingredient in a biologically active, butnon-toxic, concentration, e.g., a concentration of approximately 5 ng to50 mg of active ingredient per dosage unit (e.g., per kg subject bodyweight). The concentration utilized will be dependent upon such factorsas the overall specific biological activity of the ingredient, specificbiological activity desired, as well as the condition and body weight ofthe subject.

The pharmaceutical carrier or vehicle employed may be, for example, asolid or liquid and a variety of pharmaceutical forms may be employed.Thus, when a solid carrier is utilized, the preparation may be plainmilled, micronized in oil, tabulated, placed in a hard gelatin orenterically-coated capsule in micronized powder or pellet form, or inthe form of a troche, lozenge, or suppository. The solid carrier,containing the MNTF peptide analogue, can also be ground up prior touse.

When utilized in a liquid carrier, the preparation may be in the form ofa liquid, such as an ampule, or as an aqueous or non-aqueous liquidsuspension. For topical administration, the active ingredient may beformulated using bland, moisturizing bases, such as ointments or creams.Examples of suitable ointment bases include, but are not limited to,petrolatum plus volatile silicones, lanolin, and water in oil emulsionssuch as Eucerin® (Beiersdorf). Examples of suitable cream bases include,but are limited to, Nivea Cream® (Beiersdorf), cold cream (USP), PurposeCream® (Johnson & Johnson), hydrophilic ointment (USP), and Lubriderm®(Warner-Lambert).

Additionally, with respect to the present invention, the activeingredient may be applied internally at or near the site of the affectedmotoneuron. For example, a solid or gelled medium which is sufficientlypermeable to allow the release of the active ingredient, preferably in atimed-release manner, may be utilized for such internal application.Examples of such gels include, but are not limited to, hydrogels such aspolyacrylamide, agarose, gelatin, alginate, or other polysaccharidegums. Furthermore, the active ingredient may be imbedded in a solidmaterial, such as filter paper, which is capable of absorbing andsubsequently releasing the active ingredient, at the appropriate timeand location.

MNTF peptides according to the present invention may be provided for usein any suitable form appropriate to the protocol of administrationand/or the needs of a patient.

Apart from the pharmaceutically acceptable compositions referred toabove, the peptides may for example be provided, either singly or incombination, in lyophilized or freeze dried solid forms.

Methods of Use

Truncated MNTF molecules comprising the WMLSAFS and/or FSRYAR domain(s),such as SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, SEQ ID NO: 7, as well as other small peptide derivatives thatconstitute a “minimal” WMLSAFS and/or FSRYAR domain, are demonstratedherein to retain the neurotrophic and neurotropic function exhibited bythe MNTF1–F6 33-mer. These MNTF peptide analogues induce cell growth inneuronal cell lines by providing the same biological signal produced byhigh level expression of MNTF1 (which has been shown to selectivelypromote motor neuron viability and axonal regeneration in vitro and invivo). Such agents comprise a novel class of neurotrophic andneurotropic drug.

MNTF1 and/or its peptide analogues promote the survival of mammalianmotor neurons in vitro and stimulate proliferation of the VSC4.1 cellline, a hybrid between motor neurons and neuroblastoma cells.Accordingly, the present invention provides for the use of an MNTFpeptide analogue as a growth factor/supplement for neuronal cellcultures, including a method for promoting the survival of primarycultures of neurons or stimulating cell proliferation of neuronal celllines, by cultivating neuronal cells in vitro with an effective amountof a MNTF peptide analogue as defined above.

In vivo administration of MNTF1 to surgically axotomized rat peripheralnerves resulted in a markedly higher percentage of surviving motorneurons than untreated controls, which could be blocked byco-administration of anti-MNTF1 monoclonal antibody. Further beneficialeffects of MNTF1 were demonstrated in rats subjected to spinal cordhemi-section, repaired by a peripheral nerve autograft and implantedwith MNTF1-containing gel sections in close proximity to the nerve graftjunctions with spinal cord. MNTF1 treated animals exhibited greaternumbers of surviving motor neurons as well as improved recovery of motorand sensory function. Moreover, as demonstrated in the femoral nervemodel, described in greater detail in the examples below, treatment oftransected and sutured rat femoral nerves with MNTF1 peptides resultedin significant increases in correct projections of motor neurons totarget muscle tissues in vivo, as well as marked reductions in thenumber of incorrect projections to skin. Thus the MNTF1 peptides of thepresent invention are capable of promoting selective reinnervation oftarget muscle tissue.

Accordingly, the present invention provides therapeutic or prophylacticmethods for treating damaged or diseased motoneurons, or pathologicalconditions, such as neurodegenerative disease, and the like, which areaccomplished by the administration of an effective amount of atherapeutic agent capable of specifically promoting neuronal cellviability and/or axonal regeneration. Therapeutic or prophylacticindications can include treatment of (prevention and/or reduction of theseverity) of neurological conditions including:

a) acute, subacute, or chronic injury to the nervous system, includingtraumatic injury, chemical injury, vascular injury and deficits (such asthe ischemia resulting from stroke), together withinfectious/inflammatory and tumor-induced injury,

b) aging of the nervous system,

c) chronic immunological diseases of the nervous system or affecting thenervous system, including multiple sclerosis,

d) chronic neurodegenerative diseases of the nervous system andmusculoskeletal disorders including hereditary motoneuron diseases suchas amyotrophic lateral sclerosis, spinal muscular atrophy;

e) peripheral nerve, spinal cord and head injuries,

f) peripheral neuropathy, diabetic peripheral neuropathy, peripheralneuropathy resulting from AIDS, peripheral neuropathy resulting fromradiation treatment for cancer,

Administration of MNTF1 has also been associated with reduced scarformation and inflammation following surgical incision and repair of ratspinal cords. Moreover, recombinant MNTF1 was associated with a markedreduction in the growth of non-neuronal cells, e.g. glial cells andfibroblasts, in spinal cord explants in vitro. Thus in another aspectthe present invention provides novel MNTF peptide analogues andcompositions consisting of or containing them for use asantiproliferative agents, particularly anti-inflammatory or antifibroticagents. Moreover the present invention also provides a method ofinhibiting proliferation and or migration of non-neuronal cells,particularly fibroblasts and inflammatory cells, by administration of anMNTF peptide analog to a cell culture, more particularlyhyperproliferative scar tissue or keloid fibroblasts, or to the site ofinjury and/or scarring in a mammalian host. The invention also providessuch novel MNTF peptide analogues and compositions consisting of orcontaining them for use in wound healing and cosmetic applications.

The MNTFs of the present invention can thus be readily utilized inpharmacological applications. In vivo applications includeadministration of the factors to mammalian subjects and, in particular,to human subjects. Any mode of administration that results in thedelivery of the therapeutic agent to the desired cell is contemplated aswithin the scope of the present invention. The site of administrationand cells will be selected by one of ordinary skill in the art basedupon an understanding of the particular disorder being treated.Principles of pharmaceutical dosage and drug delivery are known and aredescribed, for example, in Ansel, H. C. and Popovich, N. G.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition, Lea& Febiger, Publisher, Philadelphia, Pa. (1990).

Administration of peptides of the invention in any of the methodsdescribed herein may be via any suitable protocol. The particular modeof administration can also be readily selected by one of ordinary skillin the art and can include, for example, oral, intravenous,subcutaneous, intramuscular, etc. with the preferred mode being topicalapplication at or near the affected site. In addition, the dosage,dosage frequency, and length of course of treatment, can be determinedand optimized by one of ordinary skill in the art depending upon theparticular degenerative disorder being treated. Such administration ofpeptides of the invention is in such an amount as to give the desiredeffective result of the peptide's activity at the intended site. Thus, aquantity which constitutes an “effective” amount may depend upon variousparameters, such as body weight of the patient, degree of activityrequired, intended site of activity, severity of the condition to betreated or prevented, all of which will be well understood andappreciated by persons skilled in the art.

As used herein, the terms “administer” includes applying the purifiedpeptide to neuronal or non-neuronal cells or tissues sufficientlyproximal to the affected site such that the polypeptide is effective atpromoting the survival of mammalian neurons and/or reduced proliferationor infiltration of non-neuronal cells, such as fibroblasts orinflammatory cells.

In yet a further aspect, the present invention provides theabove-defined MNTF peptide analogues, particularly the linked peptideanalogues of the invention, for use as immunogens for the production ofpolyclonal and monoclonal antibodies to MNTF1, especially fordiagnostic, prognostic and therapeutic uses. Such methods of productionof polyclonal and monoclonal antibodies are also within the scope of theinvention.

EXAMPLES

The invention may be appreciated in certain aspects with reference tothe following examples, offered by way of illustration, not by way oflimitation.

Materials, reagents and the like to which reference is made in thefollowing examples are obtainable from commercial sources, unlessotherwise noted.

Example 1 Production Procedure for MNTF Bio Peptides

This example shows how to manufacture MNTF peptides

1.) Synthesis—All peptides were synthesized via t-Boc chemistry using aCS536 Automated Peptide Synthesizer (CS Bio Inc.). Deprotection of theBoc groups was performed using 40% TFA(trifluoracetic acid) in MethyleneChloride. Coupling reactions were performed for a period of 2 hoursusing Diiosopropylcarbodiimide(DIC). Kaiser tests (ninhydrin based) wereperformed at the completion of each coupling cycle to check couplingefficiency.

2.) The peptide was then cleaved from the resin using HF (hydrogenfluoride). After the HF reaction, the peptide was then extracted withTFA. The extracted material was then lyophilized to obtain an accurateweight prior to the purification process.

3.) The crude peptide was then loaded on to an HPLC column packed withreverse phase C18 resin. A gradient was run from Buffer A (0.1% TFA inH₂O) to Buffer B(60% Acetonitrile in 0.1% TFA/H₂O) and fractions of theeluant were collected. The resulting fractions were analyzed byanalytical HPLC and fractions containing correct material with a purityof >95% were pooled and lyophilized.

4.) The peptide was then frozen and lyophilized. After the finallyophilization process, the peptide was checked at CS Bio for HPLCpurity and mass spectral conformation.

Example 2 IN VITRO Assay of MNTF Derivatives

Introduction

Studies of the function of motor neurons have been enhanced by thedevelopment of cell lines that mimic their function. Several neuronalcell lines including sensory-F11, motor-VSC 4.1, and adrenergic-N1E-115neuroblastoma cells, as well as Schwann cells have been used as neuronalmodels for in vitro detection of programmed cell death (PCD) orapoptosis and/or inhibitory effects of sera from amyotrophic lateralsclerosis patients or diabetic patients with neuropathy on growth,proliferation, and differentiation. In particular, the VSC4.1 cell line,a hybrid between motor neurons and neuroblastoma cells developed by Dr.Stanley Appel, has been used extensively in studies of the pathogenesisof amyotrophic lateral sclerosis (Kimura F, et al. Annals of Neurology35:164–171, 1994; Smith R G, et al. Proc Natl Acad Sci U.S.A91:3393–3397, 1994; Alexianu M E, et al. J Neurochem 63:2365–2368, 1994;Appel et al. Clin Neurosci 3:368–374, 1995–1996; and Mosier D R, et al.Ann Neurol 37:102–109, 1995, which are all incorporated herein byreference). Thus, VSC4.1 cells have been useful in examining thepathogenesis of motor neuropathies and for examination of factors thatmay protect the cells from damage in the face of systemic factors.

This example shows that certain truncated MNTF peptides stimulate theproliferation of VSC4.1 cells in a manner comparable to the MNTF133-mer. These proliferative effects may be related to the ability ofMNTF to block or reverse motor nerve loss.

Methods

Studies of proliferation were performed in 96 well plates utilizing acell proliferation assay kit from Roche Diagnostics GmbH (Mannheim,Germany). VSC4.1 cells, a motor neuron/neuroblastoma cell hybrid, werecultured in DMEM with 2% FBS containing either no MNTF or a dose rangeof MNTF from 10⁻⁸ to 10⁻⁵ g/ml. Each treatment was applied in triplicatewells. The cells were cultured for 21 hours and 5-bromo-2′-deoxyuridine(BrdU) from the assay kit was added to each well. After BrdU labelingfor 3 hours, the cells were washed, fixed and dried. The cells were thenfixed and the DNA denatured to improve access for the subsequentantibody binding. POD-labeled mouse monoclonal anti-BrdU antibody fromthe assay kit was applied for 2 hours, followed by a wash. The assay wasquantitated by adding the colorimetric assay solution (tetramethylbenzidine) and reading at 450 nm on a Wallac Vector 2 plate reader.Cells in the treatment groups were quantitated by comparing to wellsplated with a gradient of cells from 0 to 20,000 cells/well on the sameplate. Data is expressed as % of control to allow for variability fromplate to plate.

Results

The following table summarizes the results of three sets of cellproliferation assays conducted on separate occasions (10-27-02, 3-28-03and 8-4-03).

TABLE 1 MNTF Lot #** M.W. Grade* Biological Function Oct. 27, 2002 33merSEQ ID NO: 1 16064A1 3706 Rg, L Effective. OD at 10 ug/ml. 33mer SEQ IDNO: 1 16062A2 3706 Rg, C Limited Efficacy. 33mer SEQ ID NO: 1 CS14563706 GLPg, L Effective. OD at 0.1 ug/ml. 13mer SEO ID NO: 6 43426 1594Rg, C Limited Efficacy. 7mer SEQ ID NO: 3 43425 841 Rg, L Effective. ODat 1 ug/ml 6mer SEQ ID NO: 2 43427 799 Rg, L Effective. OD at 10 ug/ml.11mer SEQ ID NO: 5 43428 1273 Rg, L Effective. OD at 10 ug/ml. Mar. 28,2003 33mer SEQ ID NO: 1 16064A1 3706 Rg, L Effective. OD at 100 ug/ml.33mer SEQ ID NO: 1 CS-C118=CS1456 3706 GLPg, L Effective. OD at 10ug/ml. 13mer SEQ ID NO: 6 CS-C171=CS1510 1594 GLPg, L Effective. OD at0.1 ug/ml. 7mer SEQ ID NO: 3 CS-C173=CS1511 841 GLPg, L Effective. OD at100 ug/ml. 6mer SEQ ID NO: 2 CS-C158=CS1507 799 GLPg, L Effective. OD at1 ug/ml. 11mer SEQ ID NO: 5 CS-C172=CS1509 1273 GLPg, L Effective. OD at1 ug/ml. Aug. 4, 2003 33mer SEQ ID NO: 1 CS1456=CS-C297 3706 GLPg, LEffective. OD at 0.01 ug/ml. 21mer SEQ ID NO: 7 CS1616=CS-C382 2311GLPg, L Effective. OD at 0.1 ug/ml. 10mer SEQ ID NO: 4 CS1597=CS-C3371201 GLPg, L Effective. OD at 0.01 ug/ml. *Rg = research grade (~70%purity), GLPg = GLP grade (90–99.8% purity), L = linear peptide, C =Cyclized peptide, OD = optimal dose **CS-C### = Batch number of amanufacturing process catalog number CS#### of CS Bio Inc., the GMPproduct manufacturer. Lot numbers without “CS” are research gradeproducts from Genemed Synthesis.

Referring to the earliest studies listed in Table 1, and shown in FIG.2, MNTF 33mer (research grade) alone applied to the cultures resultedmaximally in a 31.7% increase in cell proliferation of VSC4.1 motorneuron/neuroblastoma hybrid cells at a dose of 10 μg/ml. Higher doseswere not tested. Cyclized MNTF 33mer was of limited efficacy,stimulating a maximal response of 12.9% albeit at a lower dose thanMNTF, 100 ng/ml.

Of the other peptides supplied, the most effective in this assay was theGLP grade MNTF 33mer, which stimulated a maximal response of 34.3%, alsoat a dose of 100 ng/ml (FIG. 2). Of the other peptides, the MNTF 6merand 11mer were comparably effective (FIG. 2), achieving maximalresponses of 35.1% and 32.2%, respectively. These responses were alsoseen at the highest tested dose, 10 μg/ml. The MNTF 7mer was somewhatless effective with a maximal effect of 25.1% at a dose of 1 μg/ml (FIG.2), although this peptide had a broader active dose range than the otherpreparations. The cyclized MNTF 13mer peptide was the least effective ofall peptides tested, with a maximal effect of 17.3% at 1 μg/ml (FIG. 2).Moreover, at higher doses the cyclized MNTF 13mer peptide appeared toinhibit proliferation.

Although the percentage increases reported in these preliminaryexperiments were not high, this was likely due to the high rate ofproliferation of these cells under basal conditions. With that caveat,the MNTF peptide analogues appear to be relatively effective in a dosedependent manner, with the exception of cyclized versions of thepeptides. Of the peptides tested, GLP grade 33mer (SEQ ID NO:1) was themost biologically active in this assay, given the high rate ofproliferation and the low dose needed for induction. Research grade 6mer(SEQ ID NO:2) and 11mer (SEQ ID NO:5) are comparably effective, not onlyto each other but also to the MNTF 33mers. The cyclized 13mer (SEQ IDNO:4) was markedly less effective than the other derivative peptides.Moreover, cyclized MNTF 33mer peptide showed very weak proliferativeactivity at low doses.

Additional cell proliferation assays, conducted essentially as describedabove, were performed to compare the effects of several GLP grade MNTFpeptides.

The MNTF preparations utilized for this assay were:

16064A-1 33mer Research Grade CS-C118 33mer GLP Grade CS-C158 6mer GLPGrade CS-C173 7mer GLP Grade CS-C172 11mer GLP Grade CS-C171 13mer GLPGrade

As shown in Table 1 and FIG. 3, all MNTF peptide preparations exertedproliferative effects on VSC4.1 cells, although to varying degrees. Themost potent preparation across the whole dose range was the 7mer,showing a 2.5-fold increase even at the lowest dose applied, whileexerting almost a 3.5-fold increase in proliferation at the highestdose. Although the 13mer showed some efficacy at the lowest dose, thiseffect fell off at higher doses. The two 33mers, showed a steadyincrease in response with increasing dosage, with the Research Grade33mer peaking at about a 3-fold increase at the highest dose, whileGLPgrade reached a peak 3-fold response at 10 μg/ml with no furtherincrease with increasing dosage. Less robust performers in this set ofassays were the 6mer, and the 11mer. Although these preparationsstimulated almost a 2.5-fold increase in proliferation at 1 μg/ml, theperformance across the whole dose range was not as potent as the otherisoforms of MNTF.

In this second round of experiments, the assay overall was more robust,with the most active proliferative agent, the 7mer, producing a 350%increase over controls. Four of six MNTF isoforms provided stimulatedVSC4.1 cell proliferation 250% or greater. However, one of these, the 13mer, did so only at a single dose. The other two MNTF isoforms, i.e.,the 6mer and the 11mer, although they did stimulate proliferation, didso less effectively than the others. The relative efficacy would be:7mer (SEQ ID NO:3)>33mer (SEQ ID NO:1)>>6mer (SEQ ID NO:2), 11mer (SEQID NO:5)>13mer (SEQ ID NO:6).

Further cell proliferation studies were performed, essentially asdescribed above, to test the response of the motor neuron/neuroblastomacells to three new preparations of MNTF. The MNTF preparations utilizedfor these assays were:

1456 33mer 1597 10mer 1616 21mer

As shown in Table 1 and FIG. 4, all three MNTF isoforms significantlystimulated VSC4.1 cell proliferation (ANOVA, as indicated) whenexpressed as percentage increase over control. Further, the 10mer, the21mer and the 33mer significantly (p<0.05) stimulated proliferation overcontrol at specific doses. 33mer: p<0.02. 10mer: p<0.001. 21mer:p<0.002.

As in the previous experiments, the assay overall was robust. The mostactive proliferative agent appeared to be the 10mer, producingapproximately a 200% increase over controls at the three lowest dosesused (p<0.005, ANOVA). All three of these doses were significantlydifferent from control (Tukey-Kramer post-hoc analysis). The 33merstimulated cell proliferation (p<0.01, ANOVA) nearly as well as the10mer at low doses (10⁻⁹ and 10⁻⁸ g/ml significant by Tukey-Kramerpost-hoc analysis), closely approximating the response to the 10mer athigher doses. While the 21mer significantly stimulated proliferation(p<0.05, ANOVA), the increase over controls was about half that of theother two MNTF isoforms and not significantly different at any one dose,although at high doses the responses to all three were similar.

Thus, all three isoforms of MNTF significantly stimulated motor neuronproliferation, although the 10mer (SEQ ID NO:4) and 33mer (SEQ ID NO:1)were clearly superior to the 21mer (SEQ ID NO:7). While these percentageresponses seem to be somewhat lower than in the previous assays, thiscould be due to interassay variability, caused by different activitiesof different cell passages in culture.

Example 3 In Vivo Assays for Neurotropic Activity

The Femoral Nerve Model

The specificity of motor axon regeneration was investigated in the ratfemoral nerve. Proximally, at the site of nerve transection and suture,axons that contribute to both cutaneous and muscle branches interminglethroughout the nerve. As these axons regenerate, they have equal accessto neighboring motor and sensory Schwann cell tubes in the distal nervestump. This assures an element of “choice” at the axonal level.Distally, where the specificity of regeneration is assessed, axons aresegregated into terminal cutaneous and muscle branches. Motor axons arenormally found only in the muscle branch, so any motor reinnervation ofthe cutaneous branch represents a pathfinding failure. The specificityof axon regeneration is evaluated by simultaneous application ofhorseradish peroxidase (HRP) to one distal femoral branch andfluoro-gold (FG) to the other. Motor-axon regeneration is random at 3weeks, but the number of correct projections to muscle increasesdramatically at later times. Many neurons initially contain bothtracers, and thus project collaterals to both cutaneous and musclebranches. The number of these double-labeled neurons decreases withtime. Motor axon collaterals are thus pruned from the cutaneous branch,increasing the number of correct projections to muscle at the expense ofdouble-labeled neurons. A specific interaction thus occurs betweenregenerating motor axons and muscle and/or muscle nerve that we havetermed Preferential Motor Reinnervation (PMR).

MNTF Pump Experiments

In preliminary experiments we attempted to modify motor axonregeneration in the femoral nerve by pumping MNTF 33mer at 10⁻⁴ M ontothe repair site, using an Alzet osmotic pump which discharges for atleast 2 weeks. The output of the pump was sewn to muscle adjacent to thenerve repair, so that the nerve wound would be continuously bathed withthe factor. Reinnervation of the distal femoral cutaneous and musclebranches was quantified with tracers as described above.

The controls for these preliminary experiments were a group of 10 nervesthat underwent routine suture and evaluation after three weeks ofregeneration. A mean of 92 motoneurons projected correctly to muscle atthree weeks, while a larger number (mean=117) projected incorrectly toskin. However, MNTF 33mer treatment in three animals more than doubledthe number of correct projections (mean=210) while dramatically reducingthe number incorrect projections to skin (mean=31). These differenceswere highly significant in spite of the small number of animals tested.

In a follow-up experiment MNTF 33mer was administered to eighttransected and sutured rat femoral nerves at 10⁻⁴ M, essentially asdescribed above. Controls were a group of six nerves that underwentroutine suture with pumps delivering saline but no MNTF. A mean of 100motoneurons projected correctly in the six controls, whereas a mean of87 motoneurons projected incorrectly to skin. MNTF treatment once againresulted in a significant increase in correct projections (mean=173) anda marked reduction in the number of incorrect projections to skin(mean=59).

To determine if a truncated MNTF peptide could be substituted for MNTF33mer, subsequent experiments were performed, essentially as describedabove, using the MNTF 6mer FSRYAR, at 10⁻⁴ M (n=8), 10⁻⁵ M (n=8) and10⁻⁶ M (n=8), with a total of 22 saline controls for comparison. Asshown in FIG. 5, treatment with the FSRYAR peptide (SEQ ID NO:2)resulted in significant increases in correct projections at every dosetested as well as marked reductions in the number of incorrectprojections to skin. For saline controls, the mean number of cellsprojecting correctly to muscle was 85, the mean number of incorrectprojections to skin was 85 and double-labeled neurons averaged about 41.At the optimal dose of MNTF 6mer (10⁻⁴ M) the mean number of correctprojections increased to 125, incorrect projections were reduced to 46and double-labeled neurons decreased to 23. Thus the 6mer is capable ofpromoting selective reinnervation of target muscles in a manner similarto the MNTF1 33mer.

These results demonstrate a dramatic stimulation of regeneration in therat femoral nerve model using MNTF 33mer as well as the MNTF 6merpeptide, FSRYAR (SEQ ID NO:2). We are unaware of data from other modelsthat show this significant an effect. Most manipulations result inchanges of 20–30% at best, while these results show changes of 36% togreater than 100%. Such dramatic results suggest MNTF and it's peptideanalogues are among the most potent stimulators of peripheral nerveregeneration currently available.

Example 4 Production of Anti-MNTF Peptide Antibodies and their Use inImmunoassays

Rabbit polyclonal antibodies to the MNTF 33mer as well as the MNTF 6mer,FSRYAR, were produced by Harlan Bioproducts for Sciences, Inc(Indianapolis) following their standard production protocol, whichincluded conjugating the MNTF peptide with KLH before inoculation intorabbits. More MNTF peptides were conjugated to OVA for ELISA in periodictest bleeds to confirm the high titer of antibodies before productionbleeds. The polyclonal antibodies were then purified by IgG purificationand by Affinity purification.

MNTF Competitive ELISA protocols were developed by Genetel Laboratories,LLC (Madison, Wis.) for detection or measurement of MNTF peptides, e.g.,using purified anti-MNTF6mer to detect the ability of unlabeled MNTFpeptides, such as a 6mer or 10mer, to compete with their biotinylatedcounterparts for antibody binding.

Moreover, an MNTF Sandwich ELISA protocol was developed, using anti-MNTF6mer antibodies to immobilize larger MNTF peptides containing the 6merepitope, e.g. 21mers and 33mers, which where subsequently detected usingbiotinylated anti-MNTF 33mer antibody.

Although the present invention has been described in considerable detailwith reference to certain preferred versions thereof, other versions arepossible. It will be understood that the invention is capable of furthermodifications and this application is intended to cover any variations,uses, or adoptions of the invention including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains. Therefore the spirit and scope ofthe present invention should not be limited to the description of thepreferred versions contained herein.

All publications mentioned in this specification are herein incorporatedby reference, to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

1. A motoneuronotrophic factor peptide analogue selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, and SEQ ID NO:7.
 2. A composition comprising the peptide analogueof claim 1 and a carrier.
 3. A motoneuronotrophic factor peptideanalogue of claim 1 having a sequence according to SEQ ID NO:2.
 4. Amotoneuronotrophic factor peptide analogue of claim 1 having a sequenceaccording to SEQ ID NO:3.
 5. A motoneuronotrophic factor peptideanalogue of claim 1 having a sequence according to SEQ ID NO:4.
 6. Amotonenronotrophic factor peptide analogue of claim 1 having a sequenceaccording to SEQ ID NO:5.
 7. A motoneuronotrophic factor peptideanalogue of claim 1 having a sequence according to SEQ ID NO:6.
 8. Amotoneuronotrophic factor peptide analogue of claim 1 having a sequenceaccording to SEQ ID NO:7.
 9. A motoneuronotrophic factor peptideanalogue of claim 1 wherein said peptide analogue promotes the outgrowthof mammalian motorneurons.